Before anything is weighed, the peptide has to be charged

Ask a laboratory which ionisation source produced a spectrum and you learn a great deal about the spectrum before you have looked at it. Electrospray on a modern instrument will give a peptide of four thousand daltons a family of multiply charged ions and a mass accuracy in the low parts per million. A linear MALDI time-of-flight will give a single predominantly singly charged ion and an accuracy measured in hundreds of parts per million. Both can answer the question, is this the right compound. Only one of them can answer the question, is this the right compound and nothing very close to it.

The instrument does not weigh anything

It is worth being exact about what a mass spectrometer does, because the imprecision propagates. The instrument generates ions from a sample, separates them according to the ratio of their mass to their charge, and counts them at a detector. The horizontal axis of every spectrum is mass-to-charge, conventionally written m/z and expressed in thomsons or in dimensionless units depending on the vendor’s software. Nothing is weighed. Nothing is measured against a reference mass in the sense that a balance measures against a calibration weight.

What follows from this is that every molecular weight on every certificate of analysis in this market is a calculated quantity, derived from a measured m/z by assigning a charge and subtracting the mass contribution of whatever adducted to the molecule to give it that charge — usually protons, sometimes sodium, occasionally potassium or ammonium. The assignment is normally straightforward and normally correct. It is nonetheless an assignment, and when it goes wrong it goes wrong by an integer factor, which is the kind of error that produces confident nonsense rather than a plausible discrepancy.

The practical consequence for a reader is a habit: when a mass figure appears, ask what was observed and what was inferred. A report that gives both — the m/z, the charge, and the derived neutral mass — has answered the question before it was asked.

Electrospray, and why it produces a family of ions

Electrospray ionisation works by pumping a solution of the analyte through a fine capillary held at a potential of a few kilovolts relative to the instrument’s entrance. The liquid emerging from the tip forms a cone and then a jet of charged droplets. As solvent evaporates the droplets shrink, the charge density on their surfaces rises, and at the point where electrostatic repulsion exceeds surface tension they fission into smaller droplets. Repeat this enough times and what is left is a bare, charged analyte ion in the gas phase.

Because the charge is acquired in solution and retained through desolvation, a peptide with several basic residues will carry several protons, and the population of ions reaching the analyser is distributed across charge states. This is the defining characteristic of electrospray spectra and the reason they look bewildering to a first-time reader: a single pure compound produces four or five prominent peaks, none of them at the molecular weight.

The distribution is not noise. It carries information about the number of accessible basic sites and about the conformational state of the molecule in solution, and it shifts predictably with mobile-phase composition and pH. It also has a practical advantage that matters for identity work: dividing the mass by three or four brings a large peptide into the range where instruments achieve their best resolving power and accuracy.¹

MALDI, the matrix, and the singly charged ion

Matrix-assisted laser desorption ionisation takes a different route. The peptide is mixed with a large molar excess of a small organic compound that absorbs strongly at the laser wavelength — α-cyano-4-hydroxycinnamic acid and sinapinic acid are the usual choices for peptides and proteins respectively — and the mixture is dried on a metal target. A pulsed ultraviolet laser strikes the crystal, the matrix absorbs the energy, and a plume of matrix and analyte is ejected into the vacuum with the analyte largely intact and mostly singly protonated.

Two consequences follow. First, MALDI spectra are simple: one predominant ion per compound, at the molecular weight plus one proton, which makes them easy to read and easy to print on a certificate. Second, MALDI is markedly more tolerant of salts, buffers and heterogeneous samples than electrospray, which is why it survives in routine synthesis monitoring where electrospray would require a chromatographic clean-up first.

The trade-offs are equally real. The matrix produces intense chemical background below roughly a thousand daltons, which obscures small fragments. Ion yield varies between compounds and between spots on the same target, making MALDI a poor quantitative technique. And the achievable mass accuracy on a linear instrument at peptide molecular weights is measured in hundreds of parts per million unless a reflectron and delayed extraction are in use.²

Which source a laboratory chooses, and why it should say so

The choice between the two techniques is not a matter of quality but of question. A synthesis chemist watching a coupling proceed wants a fast, salt-tolerant check that the chain has grown by the expected residue, and MALDI on a bench instrument answers that in minutes. An analytical laboratory asked whether a submitted vial contains the labelled compound and nothing closely related to it needs the resolving power and the accuracy that electrospray into a high-field analyser provides, coupled to a chromatographic separation so that species which co-elute can at least be assigned to retention times.

Both appear in this market, and reports rarely distinguish them. That matters because the two techniques have different blind spots. MALDI can induce loss of labile modifications during desorption, so a phosphorylated or otherwise fragile species may be under-represented. Electrospray suppresses ionisation of some analytes in the presence of others, so a minor component of a mixture may be absent from a spectrum in which it is genuinely present.

A certificate stating the source therefore tells a reader which class of error to consider. The Journal has stopped asking suppliers for more testing and started asking them for this line instead, on the grounds that it costs nothing and changes what the existing test can be said to support.

The charge-state arithmetic, worked

For a peptide of neutral monoisotopic mass M observed as a protonated ion carrying z protons, the mass-to-charge ratio is (M + z × 1.00728) divided by z, where 1.00728 is the mass of a proton — the mass of a hydrogen atom less the mass of an electron, a distinction that matters at parts-per-million accuracy and not at all below it.

Run this for a peptide of average mass 4113.58. The singly protonated ion appears at 4114.59. The doubly protonated ion appears at 2057.80, the triply at 1372.20, the quadruply at 1029.40 and the quintuply at 823.72. All five describe the same molecule. A reader shown only the fourth of those figures, without a charge assignment, would reasonably conclude the vial contained a peptide of about a thousand daltons.

Inverting the calculation is how the neutral mass is recovered: multiply the observed m/z by the charge and subtract z proton masses. Doing this for two or three charge states from the same spectrum and finding agreement to within the instrument’s stated accuracy is the standard internal consistency check, and it is the check that catches a misassigned charge. A single m/z with a single assumed charge has no such redundancy, which is one reason electrospray with a visible charge-state envelope is more informative than a single MALDI peak even when both instruments are equally well calibrated.

The instrument classes, and what each can support

A single quadrupole mass filter provides unit resolution and mass accuracy of a few tenths of a dalton. It is entirely adequate to confirm that a sample is broadly the compound expected and to detect large modifications, and it is the analyser in most low-cost LC-MS systems. It cannot resolve an isotopic envelope at peptide molecular weights and therefore cannot assign charge from spacing.

Time-of-flight analysers separate ions by the time they take to traverse a flight tube. A linear tube gives modest resolving power; adding a reflectron and delayed extraction raises it into the tens of thousands, and modern quadrupole time-of-flight hybrids achieve low single-figure parts-per-million accuracy with routine calibration. Orbital trapping instruments measure the frequency of ion oscillation in an electrostatic field and convert it by Fourier transform, delivering resolving powers from sixty thousand to several hundred thousand and sub-part-per-million accuracy with internal calibration. Fourier-transform ion cyclotron resonance remains the highest-performing class and the least common outside academic facilities.

What this hierarchy means for a reader of certificates is that the instrument named on the document sets a ceiling on what the document can claim, independent of the laboratory’s competence. An unnamed instrument leaves that ceiling unknown, which is why the Journal now treats the absence of an instrument name as a material omission rather than a stylistic one.

A mass that matches, and the space of things it does not exclude

Suppose a laboratory reports an observed monoisotopic mass within two parts per million of the theoretical value for the labelled peptide. What has been established is that the sample contains a species whose elemental composition is either identical to the target or differs from it in a way that happens to conserve mass to within that tolerance. This is genuinely strong evidence, and it is not identity.

The set of molecules consistent with that observation includes every permutation of the target sequence, every substitution of leucine for isoleucine and vice versa, every inversion of stereochemistry at any of the chiral centres, every migration of aspartate to isoaspartate, and — at tolerances above roughly ten parts per million — every glutamine-for-lysine exchange. It also includes any unrelated molecule of coincidentally matching composition, though in practice the chromatographic retention time excludes most of those.

The proteomics literature has spent two decades formalising exactly this problem under the heading of identification confidence, developing false-discovery-rate frameworks precisely because a matching mass is a weak identifier and a matching fragmentation pattern is a strong one.³ The research-peptide trade has borrowed the instrument from that field and not the epistemology, and the result is a market in which the word confirmed is applied to the weakest available evidence.

Peptide mapping: the test that reads the chain

Peptide mapping is the standard method by which the primary structure of a peptide or protein product is verified. The material is digested with a protease of defined specificity — trypsin cleaving after lysine and arginine, Lys-C after lysine alone, chymotrypsin after aromatic residues — and the resulting fragments are separated by reversed-phase chromatography with mass detection. Each fragment’s observed mass is matched against the masses predicted from the expected sequence, and the fraction of the chain accounted for is reported as sequence coverage.

A digest that returns every predicted fragment at the predicted mass is a far stronger identity statement than an intact mass, because it constrains the order of residues in a way that an intact measurement does not: a permuted sequence generally produces different cleavage products. It is not complete on its own, because a fragment mass is subject to the same permutation ambiguity in miniature, which is why serious mapping proceeds to a second stage of mass analysis on the fragments themselves.

For synthetic peptides of thirty to forty residues, mapping is straightforward chemistry and unremarkable chromatography, and the reason it does not appear on certificates in this market is cost and turnaround rather than difficulty. The regulatory expectation for a peptide product characterised as a biotechnological article treats structural confirmation of this kind as a matter of routine rather than of specialism.⁴⁵

Sequence coverage as a reported number

Where a peptide map is performed, the headline output is a coverage figure: the percentage of residues in the expected sequence accounted for by identified fragments. Ninety-five per cent coverage sounds close to complete and is worth interrogating, because the five per cent that is missing is not randomly located. Very short fragments elute in the solvent front and are lost. Very hydrophobic fragments retain on the column. Regions between closely spaced cleavage sites produce peptides too small to identify unambiguously.

The consequence is that the uncovered fraction tends to sit in the same places for a given protease and a given sequence, which means a laboratory reporting ninety-five per cent coverage in run after run has ninety-five per cent coverage of a specific ninety-five per cent. A second digest with a different enzyme is the conventional remedy, and a report that used two orthogonal proteases is doing something a report using one cannot.

For a reader assessing a document, the useful questions are which enzyme, what coverage, and whether the uncovered residues are identified. A map that names the missing stretch has told you where the residual uncertainty lives. A map that reports a percentage alone has told you a number whose meaning depends on information it withheld — which is, in a different guise, the same complaint this department makes about purity figures reported without a gradient.

The standing rule in this department

This publication applies one rule to every identity claim it reports, and it is worth stating in isolation because it governs the rest. A mass measurement supports a statement about composition. Only a fragmentation or mapping experiment supports a statement about sequence. Where a source says identity was confirmed, we report that a mass was measured, unless we have seen evidence of the second kind.

The rule has consequences we accept. It makes our coverage read as more sceptical than the underlying documents, because the documents claim more than they establish. It occasionally irritates laboratories which have in fact done sequence-level work and have simply not printed it, and the remedy there is a two-line email which we are glad to receive. And it means we cannot describe any research-grade vial in this market as sequence-confirmed, because on the evidence available to us almost none are.

What the rule is not is an accusation. Nothing in this article suggests that vendors are selling material other than what they label, and the Journal has no evidence of that in respect of any company it covers. The claim is narrower and, we think, harder to argue with: the documentation in general circulation does not have the discriminating power that the language on it implies, and the gap between the two is where every avoidable dispute in this market begins.

If this market spent one more pound on identity, where should it go

A fair question, and the Journal’s answer has changed. Our first instinct was to argue for sequence confirmation on every lot, and the arithmetic does not support it: peptide mapping on every batch would raise the analytical cost per vial by a multiple, and the failure mode it protects against — a wholly substituted or permuted sequence — is not the one we see evidence of.

The better allocation, on our present assessment, is orthogonal. Identity by high-resolution intact mass on every lot, at a resolving power sufficient to resolve a one-dalton shift at the parent mass, with the spectrum reproduced. Sequence confirmation once per synthesis campaign rather than once per lot, on the reasoning that the sequence is a property of the process and the lot-to-lot risk is degradation rather than misconstruction. And a chromatographic method shallow enough to separate the deamidated form, because that is the change most likely to have occurred between the certificate and the buyer.

That package is not expensive. Two of the twenty companies in our dossier programme already do something close to the first item, and one has told us it is costed for the second. Whether any of it happens depends on whether buyers ever ask, which is a market question rather than a scientific one and is therefore the harder of the two.

Readers who take one thing from this piece should take the arithmetic. Isotope spacing is one over the charge. Deamidation is one dalton, oxidation is sixteen, and stereochemical inversion is nothing at all. A tolerance of ±1 dalton on a four-thousand-dalton peptide is two hundred and forty parts per million and excludes almost nothing worth excluding. Those four facts are sufficient to read most of the identity claims in circulation, and they fit on the back of an envelope.